In recent years, the progress of mass spectrometry has been striking and this method has been studied and utilized for the purpose of the detection and measurement of various biological materials. As mass spectrometers, mass spectrometers with various functions have been developed which include a mass spectrometer using electrosprayionization, a mass spectrometer having liquid chromatography-mass spectrometry (LC-MS), and a MS/MS spectrum or tandem mass spectrum in which two spectrometers are connected; a combination of these functions is utilized for the detection, measurement and quantification of biological materials (Patent Documents 1 to 3).
A technique for exhaustively analyzing a protein using a mass spectrometer is called proteomics. A protein is a macromolecule and has a three dimensional structure: thus, it is not suitable for exhaustive separation by liquid chromatography and analysis using a mass spectrometer. Therefore, generally, a protein is denatured, modified, precipitated, and then cut into peptides using a protein-digesting enzyme such as trypsin, and the peptides are separated by liquid chromatography and analyzed using a mass spectrometer. The digested peptide fragments can be identified to identify a protein present in a protein sample and a digested peptide fragment can be further quantified to quantify the expression level of a target protein in the protein sample.
The inventors have already invented methods for exhaustively and simultaneously quantifying the absolute expression levels of a membrane protein and a metabolizing enzyme using a mass spectrometer (Patent Documents 4 and 5). For conventional proteomics intended for identification, the efficiency of a pretreatment such as denaturation, modification, precipitation or digestion has presented little problem although it affects sensitivity. However, for the quantitative methods of the inventors, a sufficient pretreatment forms a precondition because the quantitative value of a targeted digested peptide is used as the quantitative value of a target protein; the quality of the efficiency of the pretreatment greatly influences the accuracy and reliability of the quantitative value. Thus, it is essential for the quantification of a protein using mass spectrometry to evaluate the efficiency of the pretreatment for each sample to confirm that the pretreatment has taken place sufficiently.
Previously, for the quantification of a protein using a mass spectrometer, although there have been findings on the preparation of a stable isotope-labeled protein having the same sequence as that of the protein to be quantified to use the stable isotope-labeled protein as an internal standard, there have been no findings on the evaluation of the efficiency of a pretreatment (Non-patent Document 1). For the evaluation of the efficiency of a pretreatment, a method using a certain purified protein already present in nature as a standard protein and one of its digested peptide fragments as an evaluation peptide has been carried out. However, the method has a problem that it affects the quantitative value and performs an overestimation when a protein partially having the same amino acid sequence as that of the evaluation peptide is present in a measurement sample. In addition, in the case of evaluation using one evaluation peptide, there is a possibility that the evaluation peptide is completely cut even when digestion does not sufficiently take place on the whole, posing a problem of overestimating the pretreatment efficiency.
There is a method which involves preparing an artificial protein labeled with a stable isotope by producing an artificial protein in which a target digested peptide used for the quantification of each protein is incorporated and culturing Escherichia coli in a medium containing an amino acid labeled with a stable isotope. A method is reported which involves adding the artificial protein labeled with a stable isotope to a protein to be quantified, pretreating the mixture, measuring the peak area ratio (of the non-labeled target digested peptide/the stable isotope-labeled target digested peptide) using a mass spectrometer, and calculating the relative expression ratio of each protein corrected for the pretreatment efficiency from the peak area ratio (Non-patent Document 2). However, this method limits the target protein to be quantified, requires that the artificial protein be redesigned and repurified after each change of the target protein, and therefore has a large problem in general versatility.